I. Field of the Invention
This invention is directed to a method of enhancing the biological activity of vascular endothelial growth factors (VEGF). The invention further concerns certain VEGF variants having enhanced biological activity. The invention also concerns methods and means for preparing these variants, and pharmaceutical compositions comprising them. The invention further concerns methods of treatment using, and articles of manufacture containing such VEGF variants.
II. Description of Background and Related Art
Vascular endothelial growth factor (VEGF), also referred to as vascular permeability factor (VPF), is a secreted protein generally occurring as a homodimer and having multiple biological functions. The native human VEGF monomer occurs as one of five known isoforms, consisting of 121, 145, 165, 189, and 206 amino acid residues in length after removal of the signal peptide. The corresponding homodimer isoforms are generally referred to as hVEGF121, hVEGF145, hVEGF165, hVEGF189, and hVEGF206, respectively. The known isoforms are generated by alternative splicing of the RNA encoded by a single human VEGF gene that is organized in eight exons, separated by seven introns, and has been assigned to chromosome 6p21.3 (Vincenti et al., Circulation 93:1493-1495 [1996]). A schematic representation of the various forms of VEGF generated by alternative splicing of VEGF mRNA is shown in FIG. 1, where the protein sequences encoded by each of the eight exons of the VEGF gene are represented by numbered boxes. VEGF165 lacks the residues encoded by exon 6, while VEGF121 lacks the residues encoded by exons 6 and 7. With the exception of hVEGF121, all VEGF isoforms bind heparin. The lack of a heparin-binding region in hVEGF121 is believed to have a profound effect on its biochemical properties. In addition, proteolytic cleavage of hVEGF produces a 110-amino acid species (hVEGF110).
hVEGF121 and hVEGF165 are the most abundant of the five known isoforms. They both bind to the receptors KDR/Flk-1 and Flt-1 but hVEGF165 additionally binds to a more recently discovered receptor (VEGF165R) (Soker et al., J. Biol. Chem. 271:5761-5767 [1996]). VEGF165R has been recently cloned by Soke et al., and shown to be equivalent to a previously-defined protein known as neuropilin-1 (Cell 92:735-745 [1998]). The binding of hVEGF165 to the latter receptor is mediated by the exon-7 encoded domain, which is not present in hVEGF121.
VEGF is a potent mitogen for micro- and macrovascular endothelial cells derived from arteries, veins, and lymphatics, but shows significant mitogenic activity for virtually no other normal cell types. The denomination of VEGF reflects this narrow target cell specificity. VEGF has been shown to promote angiogenesis in various in vivo models, including, for example, the chick chorioallantoic membrane (Leung et al., Science 246:1306-1309 [1989]; Plouet et al., EMBO J 8:3801-3806 [1989]); the rabbit cornea (Phillips et al., In Vivo 8:961-965 [1995]); the primate iris (Tolentino et al, Arch Opthalmol 114:964-970 [1996]); and the rabbit bone (Connolly et al, J. Clin. Invest. 84:1470-1478 [1989]). As a result of its pivotal role in angiogenesis (spouting of new blood vessels) and vascular remodeling (enlargement of preexisting vessels), VEGF is a promising candidate for the treatment of coronary artery disease and peripheral vascular disease. High levels of VEGF are expressed in various types of tumors in response to tumor-induced hypoxia (Dvorak et al., J. Exp. Med. 174:1275-1278 [1991]; Plate et al., Nature 359:845-848 [1992]), and tumor growth has been inhibited by anti-VEGF antibodies and soluble VEGF receptors (Kim et al, Nature 362:841-844 [1993]; Kendall and Thomas, PNAS USA 90:10705-10709 [1993]).
The biologically active form of hVEGF121 is a homodimer (in which the two chains are oriented anti-parallel) containing one N-linked glycosylation site per monomer chain at amino acid position 75 (Asn-75), which corresponds to a similar glycosylation site at position 75 of hVEGF165. If the N-linked glycosylation structures are removed, the biologically active molecule has a molecular weight of about 28 kDa with a calculated pI of 6.1. Each monomer chain in the hVEGF121 homodimer has a total of nine cysteines, of which six are involved in the formation of three intra-chain disulfides stabilizing the monomeric structure, two are involved in two inter-chain disulfide bonds stabilizing the dimeric structure, while until recently one cysteine (Cys-116) has been believed to remain unpaired. Recently, a Cys(116)-Cys(116) inter-chain disulfide bond has been reported in E. coli derived recombinant hVEGF121 (Keck et al., Arch. Biochem. Biophys. 344:103-113 [1997]), and there are data indicating that VEGF121 as produced in nature, also occurs in the form of homodimers that have the cysteines at positions 116 disulfide-bonded with each other. EP 0 484 401 describes the substitution of one or more cysteine residues, including Cys-116, within the native VEGF molecule by another amino acid, to render the molecule more stable.